Assembling assays into a master MultiAssayExperiment object

library(tidyverse)
library(magrittr)
library(gt)
library(patchwork)
library(SummarizedExperiment)
library(tidySummarizedExperiment)
library(MultiAssayExperiment)

tmp <- fs::dir_map("R scripts/", source)

theme_set(theme_light())

Clinical data (CRF data)

CRF data (raw and QCed)

crf_files <- 
  get_01_output_dir() |> 
  fs::dir_ls() |> 
  str_subset("/01_")

crf_clean_file <- crf_files |> str_subset("01_crf_clean") |> sort(decreasing = TRUE) |> magrittr::extract(1)
crf_raw_file <- crf_files |> str_subset("01_crf_raw") |> sort(decreasing = TRUE) |> magrittr::extract(1)
crf_dict_file <- crf_files |> str_subset("01_crf_plates_dictionary") |> sort(decreasing = TRUE) |> magrittr::extract(1)

load(crf_clean_file, verbose = TRUE)

Loading objects:
  crf_clean

load(crf_raw_file, verbose = TRUE)

Loading objects:
  crf_raw

load(crf_dict_file, verbose = TRUE)

Loading objects:
  crf_plates_dictionary

rm(crf_clean_file, crf_raw_file, crf_dict_file)

Participants table

load(
  crf_files |> str_subset("01_participants_2025") |> sort(decreasing = TRUE) |> magrittr::extract(1),
  verbose = TRUE
  )

Loading objects:
  participants

load(
  crf_files |> str_subset("01_participants_variable_dictionary") |> sort(decreasing = TRUE) |> magrittr::extract(1),
  verbose = TRUE
  )

Loading objects:
  participants_variable_dictionary

load(
  crf_files |> str_subset("01_participant_crfs_merged") |> sort(decreasing = TRUE) |> magrittr::extract(1),
  verbose = TRUE
  )

Loading objects:
  participant_crfs_merged

Visits table

load(
  crf_files |> str_subset("01_visits_2025") |> sort(decreasing = TRUE) |> magrittr::extract(1),
  verbose = TRUE
  )

Loading objects:
  visits

load(
  crf_files |> str_subset("01_visits_long_2025") |> sort(decreasing = TRUE) |> magrittr::extract(1),
  verbose = TRUE
  )

Loading objects:
  visits_long

load(
  crf_files |> str_subset("01_visits_crfs_merged") |> sort(decreasing = TRUE) |> magrittr::extract(1),
  verbose = TRUE
  )

Loading objects:
  visits_crfs_merged

visits |> 
  ggplot() +
  aes(x = study_day, y = pid |> factor(), color = visit_code |> factor()) +
  geom_point() 

We define the uid (unique identifier):

visits <- 
  visits |> 
  mutate(
    uid = str_c(pid, visit_code, sep = "_")
  ) |> 
  select(uid, everything())

Exposures table

load(
  crf_files |> str_subset("01_exposures_") |> sort(decreasing = TRUE) |> magrittr::extract(1),
  verbose = TRUE
  )

Loading objects:
  exposures

Assays: loading the SummarizedExperiment objects

data_dir <- get_01_output_dir()
se_files <- fs::dir_ls(data_dir, regexp = "se_.*\\.rds$") |> sort(decreasing = TRUE)

Metagenomics data

mg_file <- se_files[grepl("mg", se_files)][1]
se_mg <- readRDS(mg_file)
se_mg <- check_se(se_mg)
se_mg

# A SummarizedExperiment-tibble abstraction: 775,884 × 58
# Features=779 | Samples=996 | Assays=count, count_corr, rel_ab_all,
#   rel_ab_bact, rel_ab
   .feature .sample  count count_corr rel_ab_all rel_ab_bact rel_ab uid   mg_uid
   <chr>    <chr>    <dbl>      <dbl>      <dbl>       <dbl>  <dbl> <chr> <chr> 
 1 C0022A1  0681000…     0          0          0           0      0 0681… MG_202
 2 C0059E1  0681000…     0          0          0           0      0 0681… MG_202
 3 C0175A1  0681000…     0          0          0           0      0 0681… MG_202
 4 FF00018  0681000…     0          0          0           0      0 0681… MG_202
 5 FF00051  0681000…     0          0          0           0      0 0681… MG_202
 6 UC101    0681000…     0          0          0           0      0 0681… MG_202
 7 122010   0681000…     0          0          0           0      0 0681… MG_202
 8 185329   0681000…     0          0          0           0      0 0681… MG_202
 9 C0006A1  0681000…     0          0          0           0      0 0681… MG_202
10 C0028A1  0681000…     0          0          0           0      0 0681… MG_202
# ℹ 40 more rows
# ℹ 49 more variables: sample_type <fct>, control_type <fct>,
#   total_non_human_reads <dbl>, rel_ab_multi_genera <dbl>,
#   poor_quality_mg_data <lgl>, cst <chr>, sub_cst <chr>, valencia_score <dbl>,
#   swab_barcode <chr>, mg_pid <chr>, mg_visit_code <chr>,
#   visit_code_flagged <lgl>, mg_sample_type <chr>, ext_lib_plate_nb <dbl>,
#   ext_lib_plate_id <dbl>, ext_lib_position <chr>, …

qPCR data

qPCR_file <- se_files[grepl("pcr_agg", se_files)][1]
se_qPRC <- readRDS(qPCR_file)
se_qPRC <- check_se(se_qPRC)
se_qPRC

# A SummarizedExperiment-tibble abstraction: 16,464 × 23
# Features=16 | Samples=1029 | Assays=n_non_na, dilution, copies_per_swab_med,
#   copies_per_swab_mean, copies_per_swab_cv
   .feature .sample   n_non_na dilution copies_per_swab_med copies_per_swab_mean
   <chr>    <chr>        <int>    <dbl>               <dbl>                <dbl>
 1 C0175A1  06810000…        3        5                   0                    0
 2 C0022A1  06810000…        3        5                   0                    0
 3 C0059E1  06810000…        3        5                   0                    0
 4 UC101    06810000…        3        5                   0                    0
 5 FF00018  06810000…        3        5                   0                    0
 6 FF00051  06810000…        3        5                   0                    0
 7 185329   06810000…        3       20                   0                    0
 8 C0028A1  06810000…        3       20                   0                    0
 9 C0112A1  06810000…        3       20                   0                    0
10 FF00004  06810000…        3       20                   0                    0
# ℹ 310 more rows
# ℹ 17 more variables: copies_per_swab_cv <dbl>, uid <chr>,
#   qpcr_sample_type <fct>, sample_type <chr>, control_type <chr>,
#   qpcr_sample_id <chr>, vibr_sample_id <chr>, ext_lib_plate_nb <chr>,
#   target <fct>, fluor <chr>, strain_group_qpcr <chr>, taxon_label <chr>,
#   LBP <fct>, strain_id <fct>, strain_origin <fct>, biose_id <chr>,
#   pcr_plate_ids <chr>

16S rRNA amplicon sequencing

amplicon_file <- se_files[grepl("16S_agg", se_files)][1]
se_16S <- readRDS(amplicon_file)
se_16S <- check_se(se_16S)
se_16S

# A SummarizedExperiment-tibble abstraction: 3,365,223 × 48
# Features=817 | Samples=4119 | Assays=counts, rel_ab
   .feature  .sample counts rel_ab uid   exclude_sample total_counts sample_type
   <chr>     <chr>    <dbl>  <dbl> <chr> <chr>                 <dbl> <chr>      
 1 Lactobac… 068100…   6904 0.0933 0681… No                    74008 Clinical s…
 2 Lactobac… 068100…      0 0      0681… No                    74008 Clinical s…
 3 Gardnere… 068100…   8284 0.112  0681… No                    74008 Clinical s…
 4 Ca_Lachn… 068100…      0 0      0681… No                    74008 Clinical s…
 5 Sneathia… 068100…  21143 0.286  0681… No                    74008 Clinical s…
 6 Fannyhes… 068100…   1601 0.0216 0681… No                    74008 Clinical s…
 7 Sneathia… 068100…      0 0      0681… No                    74008 Clinical s…
 8 Prevotel… 068100…   1963 0.0265 0681… No                    74008 Clinical s…
 9 Prevotel… 068100…      0 0      0681… No                    74008 Clinical s…
10 f_Dethio… 068100…   3629 0.0490 0681… No                    74008 Clinical s…
# ℹ 40 more rows
# ℹ 40 more variables: control_type <chr>, sample_id <chr>,
#   sample_id_barcode <chr>, patient_id <chr>, visit_code_ps <chr>,
#   idt_adapter_name <chr>, i5_index_forward <chr>, i5_index_reverse <chr>,
#   i7_index_reverse <chr>, i7_index <chr>, sample_id_2 <chr>,
#   total_spike_in_rel <dbl>, total_spike_in_counts <dbl>, cst <chr>,
#   sub_cst <chr>, score <dbl>, specimen_type <chr>, well_id <chr>, …

Luminex data

luminex_file <- se_files[grepl("luminex_agg", se_files)][1]
se_luminex <- readRDS(luminex_file)
se_luminex <- check_se(se_luminex)
se_luminex

# A SummarizedExperiment-tibble abstraction: 40,800 × 37
# Features=48 | Samples=850 | Assays=obs_conc, obs_conc_imp, value_type,
#   value_types, sd_log10_obs_conc_imp, obs_conc_log10, obs_conc_imp_log10
   .feature  .sample        obs_conc obs_conc_imp value_type         value_types
   <chr>     <chr>             <dbl>        <dbl> <chr>              <chr>      
 1 CTACK     068100004_0000    207.         207.  Observed concentr… Observed c…
 2 Eotaxin   068100004_0000     90.7         90.7 Observed concentr… Observed c…
 3 FGF basic 068100004_0000    526.         526.  Observed concentr… Observed c…
 4 G-CSF     068100004_0000    767.         767.  Observed concentr… Observed c…
 5 GM-CSF    068100004_0000     64.2         64.2 Observed concentr… Observed c…
 6 GRO-a     068100004_0000   3001.        3001.  Observed concentr… Observed c…
 7 HGF       068100004_0000   3805.        3805.  Observed concentr… Observed c…
 8 IFN-a2    068100004_0000    328.         328.  Observed concentr… Observed c…
 9 IFN-g     068100004_0000   4074.        4074.  Observed concentr… Observed c…
10 IL-10     068100004_0000     33.2         33.2 Observed concentr… Observed c…
# ℹ 38 more rows
# ℹ 31 more variables: sd_log10_obs_conc_imp <dbl>, obs_conc_log10 <dbl>,
#   obs_conc_imp_log10 <dbl>, uid <chr>, sample_id <chr>, visit <dbl>,
#   weight <dbl>, volume <dbl>, approx_volume_based_on_aliquot_repos <dbl>,
#   aliquot_volume <dbl>, dilution_200ul_tube <dbl>,
#   volume_added_for_assay <dbl>, dilution_manifest <dbl>, dilution_new <dbl>,
#   shorthand_title <chr>, alternate_title <chr>, …

Flow cytometry data

flow_file <- se_files[grepl("flow", se_files)][1]
se_flow <- readRDS(flow_file)
se_flow <- check_se(se_flow)
se_flow

# A SummarizedExperiment-tibble abstraction: 3,916 × 15
# Features=11 | Samples=356 | Assays=count, percentage
   .feature     .sample        count percentage uid   sample machine sample_type
   <chr>        <chr>          <dbl>      <dbl> <chr> <chr>  <chr>   <chr>      
 1 Live         068100004_10… 2.16e6      NA    0681… Speci… 5R      Clinical s…
 2 Granulocytes 068100004_10… 2.98e4       1.38 0681… Speci… 5R      Clinical s…
 3 B_cells      068100004_10… 3.68e2      NA    0681… Speci… 5R      Clinical s…
 4 APC          068100004_10… 7.88e2      NA    0681… Speci… 5R      Clinical s…
 5 CD40         068100004_10… 4.06e2      51.5  0681… Speci… 5R      Clinical s…
 6 CD80         068100004_10… 3.82e2      48.5  0681… Speci… 5R      Clinical s…
 7 CD86         068100004_10… 4.92e2      62.4  0681… Speci… 5R      Clinical s…
 8 CD4          068100004_10… 4.13e3      NA    0681… Speci… 5R      Clinical s…
 9 CD8          068100004_10… 1.48e3      NA    0681… Speci… 5R      Clinical s…
10 DblePosCD4   068100004_10… 1.58e2       3.82 0681… Speci… 5R      Clinical s…
# ℹ 210 more rows
# ℹ 7 more variables: control_type <chr>, pid_assay <chr>,
#   visit_code_assay <chr>, site_assay <chr>, cell_type <chr>,
#   parent_cell_type <fct>, cell_type_label <fct>

Creating the MultiAssayExperiment object

Assay list

assay_list <- 
  bind_rows(
    tibble(
      mae_assay_name = "mg", 
      se_object_name = "se_mg", 
      assay_label = "Metagenomic"
      ),
    tibble(
      mae_assay_name = "qPCR", 
      se_object_name = "se_qPRC", 
      assay_label = "qPCR"
      ),
    tibble(
      mae_assay_name = "amplicon", 
      se_object_name = "se_16S", 
      assay_label = "16S rRNA amplicon seq."
      ),
    tibble(
      mae_assay_name = "luminex", 
      se_object_name = "se_luminex", 
      assay_label = "Luminex"
      ),
    tibble(
      mae_assay_name = "flow", 
      se_object_name = "se_flow", 
      assay_label = "Flow cytometry"
      )
  ) |> 
  mutate(
    assay_label = assay_label |> fct_inorder()
  )

We will assemble the following tables (SE objects):

assay_list |> gt()

mae_assay_name	se_object_name	assay_label
mg	se_mg	Metagenomic
qPCR	se_qPRC	qPCR
amplicon	se_16S	16S rRNA amplicon seq.
luminex	se_luminex	Luminex
flow	se_flow	Flow cytometry

mae_coldata_assays tables

available_samples <- 
  map(
    1:nrow(assay_list),
    function(i, assay_list){
      se <- get(assay_list$se_object_name[i])
      tibble(
        assay = assay_list$mae_assay_name[i],
        se_colname = se |> colnames(), 
        uid = se$uid, 
        sample_type = se$sample_type,
        control_type = se$control_type,
        )
    },
    assay_list = assay_list
  ) |> 
  bind_rows()

if (!all(available_samples$se_colname == available_samples$uid, na.rm = TRUE)) {
  stop("The uid column in the SE objects does not match their colnames")
}

mae_coldata_assays <-
  available_samples |>
  group_by(uid) |>
  summarize(
    n_sample_type = n_distinct(sample_type),
    n_control_type = n_distinct(control_type),
    sample_type = sample_type[1],
    control_type = control_type[1],
    assays = str_c(assay, collapse = ", ")
  )

if (any(mae_coldata_assays$n_sample_type > 1)) {
  warning("Some samples have multiple sample types")
}

if (any(mae_coldata_assays$n_control_type > 1)) {
  warning("Some samples have multiple control types")
}

mae_coldata_assays <- 
  mae_coldata_assays |> 
  select(uid, sample_type, control_type, assays) 

The mae_coldata_assays table contains the following columns:

mae_coldata_assays |> colnames()

[1] "uid"          "sample_type"  "control_type" "assays"      

mae_coldata_assays |> str()

tibble [4,184 × 4] (S3: tbl_df/tbl/data.frame)
 $ uid         : chr [1:4184] "068100004_0000" "068100004_1000" "068100004_1001" "068100004_1002" ...
 $ sample_type : chr [1:4184] "Clinical sample" "Clinical sample" "Clinical sample" "Clinical sample" ...
 $ control_type: chr [1:4184] "" "" "" "" ...
 $ assays      : chr [1:4184] "mg, qPCR, amplicon, luminex" "mg, qPCR, amplicon, luminex, flow" "amplicon" "amplicon" ...

mae_coldata_assays |> 
  dplyr::count(sample_type, control_type) |> 
  gt("Number of samples per sample type and control type") 

sample_type	control_type	n
Clinical sample		3962
Clinical sample (other study)		7
Negative control	Nuclease-free water	46
Negative control	Unused swab + C2	47
Positive control	Mock 1	57
Positive control	Mock 2	57
Test sample		8

We also remove the columns sample_type and control_type from the colData of the individual assays to avoid conflicts when joining with the mae@colData table in downstream analyses.

for (i in 1:nrow(assay_list)) {
  se <- get(assay_list$se_object_name[i])
  if (("sample_type" %in% colnames(colData(se))) | ("control_type" %in% colnames(colData(se)))) {
    colData(se) <- colData(se)[, !colnames(colData(se)) %in% c("sample_type", "control_type")]
  }
  assign(assay_list$se_object_name[i], se)
}

MAE@colData: joining the visits and mae_coldata_assays tables

We first do a full join of the visits and mae_coldata_assays tables by uid. This will allow us to keep all visits, even those that do not have assay data, and to identify which visits are in the CRF data and which are in the assay data.

mae_coldata <- 
  # we first do a full join of the CRF visits and mae_coldata_assays by uids;
  dplyr::full_join(
    visits |> select(-starts_with("specimen_collection")) |> mutate(in_CRF = TRUE),
    mae_coldata_assays |> mutate(in_assays = TRUE) |> select(in_assays, everything()),
    by = join_by(uid)
  ) |> 
  mutate(
    in_CRF = in_CRF |> replace_na(FALSE),
    in_assays = in_assays |> replace_na(FALSE)
  )

# We notice that there are 5 "Clinical sample" entries for which we do have assay data but that did not appear in the CRF data:

mae_coldata |> 
  filter(is.na(visit_code), sample_type == "Clinical sample") 
  
# we fix these below

mae_coldata <- 
  mae_coldata |> 
  mutate(
    needs_fixing = is.na(pid) & (sample_type == "Clinical sample") & str_detect(uid, "[0-9]{9}_[0-9]{4}"),
    pid = 
      case_when(
        needs_fixing ~ str_remove(uid, "_[0-9]{4}"),
        TRUE ~ pid
      ),
    visit_code =
      case_when(
        needs_fixing ~ str_remove(uid, "[0-9]{9}_"),
        TRUE ~ visit_code
      ),
    study_day = 
      case_when(
        needs_fixing & (visit_code %in% c("0001", "0011")) ~ -7,
        needs_fixing & in_assays & (visit_code == "1000") ~ 0,
        TRUE ~ study_day
      ),
    visit_attended = 
      case_when(
        is.na(visit_attended) & needs_fixing ~ TRUE,
        TRUE ~ visit_attended
      ),
    visit_planned = 
      case_when(
        is.na(visit_planned) & needs_fixing ~ "Unplanned visit",
        TRUE ~ visit_planned
      ),
    visit_type = 
      case_when(
        is.na(visit_type) & needs_fixing ~ "Clinic",
        TRUE ~ visit_type
      )
  ) 

We then merge that table with the participants table to add the participant-invariant information (e.g., randomized, mITT, etc.) to the mae_coldata table.

# we add the participant-invariant information from the `participants` table
mae_coldata <- 
  mae_coldata |> 
  dplyr::left_join(
    participants,
    by = join_by(pid)
  )

mae_coldata <- mae_coldata |> select(-needs_fixing)

mae_coldata |> 
  dplyr::count(in_CRF, randomized, in_assays, sample_type, assays) |> 
  gt()

in_CRF	randomized	in_assays	sample_type	assays	n
FALSE	FALSE	TRUE	Clinical sample	amplicon	1
FALSE	FALSE	TRUE	Clinical sample	mg, qPCR, amplicon, flow	2
FALSE	NA	TRUE	Clinical sample (other study)	mg, qPCR	7
FALSE	NA	TRUE	Negative control	amplicon	70
FALSE	NA	TRUE	Negative control	mg, qPCR	12
FALSE	NA	TRUE	Negative control	qPCR	11
FALSE	NA	TRUE	Positive control	amplicon	91
FALSE	NA	TRUE	Positive control	mg	1
FALSE	NA	TRUE	Positive control	mg, qPCR	10
FALSE	NA	TRUE	Positive control	qPCR	12
FALSE	NA	TRUE	Test sample	qPCR	8
TRUE	FALSE	FALSE	NA	NA	414
TRUE	FALSE	TRUE	Clinical sample	mg, qPCR, amplicon	66
TRUE	FALSE	TRUE	Clinical sample	mg, qPCR, amplicon, luminex	5
TRUE	FALSE	TRUE	Clinical sample	qPCR, amplicon	1
TRUE	TRUE	FALSE	NA	NA	864
TRUE	TRUE	TRUE	Clinical sample	amplicon	2989
TRUE	TRUE	TRUE	Clinical sample	amplicon, luminex	1
TRUE	TRUE	TRUE	Clinical sample	flow	1
TRUE	TRUE	TRUE	Clinical sample	luminex, flow	1
TRUE	TRUE	TRUE	Clinical sample	mg, qPCR, amplicon	49
TRUE	TRUE	TRUE	Clinical sample	mg, qPCR, amplicon, flow	3
TRUE	TRUE	TRUE	Clinical sample	mg, qPCR, amplicon, luminex	491
TRUE	TRUE	TRUE	Clinical sample	mg, qPCR, amplicon, luminex, flow	348
TRUE	TRUE	TRUE	Clinical sample	mg, qPCR, luminex	2
TRUE	TRUE	TRUE	Clinical sample	qPCR, amplicon, luminex	1
TRUE	TRUE	TRUE	Clinical sample	qPCR, amplicon, luminex, flow	1

Imputing study_day for daily visit_code that did not have CRF data

There were a few daily swabs with daily visit_code that did not have CRF data, and consequently did not have study days assigned.

We impute the missing values using the study_days from “surrounding” visits.

# study_days_for_daily_codes <- 
#   tibble(study_day = (1:(7*6)) -1) |> 
#   mutate(
#     visit_code = 1000 + ((study_day %% 7) + 1) + (study_day %/% 7) * 100,
#     visit_code = visit_code |> as.character()
#   ) |> 
#   dplyr::rename(exp_study_day = study_day) |> 
#   select(visit_code, exp_study_day) 

study_days_for_daily_codes <- 
  mae_coldata |> 
  dplyr::filter(visit_type == "Home") |> 
  dplyr::count(visit_code, study_day) |> 
  arrange(visit_code, -n) |> 
  group_by(visit_code) |> 
  slice_head(n = 1) |> 
  ungroup() |> 
  dplyr::rename(exp_study_day = study_day) |> 
  select(-n)

study_days_for_daily_codes |> 
  ggplot(aes(x = exp_study_day, y = visit_code)) +
  geom_point()

study_days_for_daily_codes <-
  study_days_for_daily_codes |> 
  mutate(
    exp_study_day = 
      case_when(
        is.na(exp_study_day) & (visit_code %in% c(1501:1503)) ~ as.numeric(visit_code) - 1501 + 36,
        TRUE ~ exp_study_day
      )
  )

pid_with_missing_daily_study_days <- 
  mae_coldata |> 
  filter(is.na(study_day), in_assays) |> 
  select(pid) |> distinct() |> 
  left_join(mae_coldata, by = join_by(pid)) |> 
  arrange(pid, visit_code) |> 
  left_join(study_days_for_daily_codes) |> 
  mutate(diff = study_day - exp_study_day) |>
  group_by(pid) |> 
  fill(diff, .direction = "downup") |> 
  ungroup() |> 
  mutate(
    fixed_study_day = 
      case_when(
        is.na(study_day) & !is.na(exp_study_day) ~ exp_study_day + diff,
        TRUE ~ study_day
      )
  ) |> 
  select(uid, pid, visit_code, study_day, fixed_study_day, exp_study_day, diff, in_CRF, in_assays, crf_plates, assays)

# pid_with_missing_daily_study_days |> View()

mae_coldata <- 
  mae_coldata |> 
  left_join(pid_with_missing_daily_study_days |> select(uid, fixed_study_day), by = join_by(uid)) |> 
  mutate(
    study_day =
      case_when(
        is.na(study_day) ~ fixed_study_day,
        TRUE ~ study_day
      )
  ) |> 
  select(-fixed_study_day)

mae_coldata |> 
  filter(is.na(study_day), in_assays) |> 
  View()

# all is fixed

Creating the visit, visit_number, and study_week columns

visit_dict <- 
  bind_rows(
    tibble(
      visit = "screening", 
      definition = "Screening visit for which we have the most assay data. If two screening visits have the same number of assays, we take the last one.", 
      possible_visit_codes = "0000, 0001, 0010, 0011",
      visit_number = "V0",
      study_week = -1
    ),
    tibble(
      visit = "add. screening", 
      definition = 'If a participant has data for two screening visits, one is the "main" screening visit, and the other is labelled as an additional screening visit.', 
      possible_visit_codes = "0000, 0001, 0010, 0011",
      visit_number = "V0b",
      study_week = -2
      ),
    tibble(
      visit = "pre-MTZ", 
      definition = "Visit before the metronidazole treatment (MTZ) starts.", 
      possible_visit_codes = "1000, 1011",
      visit_number = "V1",
      study_week = 0
      ),
    tibble(
      visit = "post-MTZ", 
      definition = "Visit after the metronidazole treatment (MTZ) ends.", 
      possible_visit_codes = "1100",
      visit_number = "V2",
      study_week = 1
      ),
    tibble(
      visit = "post-INT", 
      definition = "Visit after the intervention (INT) ends.", 
      possible_visit_codes = "1200, 1211",
      visit_number = "V3",
      study_week = 2
      ),
    tibble(
      visit = "1w post-INT", 
      definition = "Visit one week after the intervention (INT) ends.", 
      possible_visit_codes = "1300",
      visit_number = "V4",
      study_week = 3
      ),
    tibble(
      visit = "2w post-INT", 
      definition = "Visit two weeks after the intervention (INT) ends.", 
      possible_visit_codes = "1400",
      visit_number = "V5",
      study_week = 4
      ),
    tibble(
      visit = "3w post-INT", 
      definition = "Visit three weeks after the intervention (INT) ends.", 
      possible_visit_codes = "1500, 1511, 1512",
      visit_number = "V6",
      study_week = 5
      ),
    tibble(
      visit = "5w post-INT", 
      definition = "Visit five weeks after the intervention (INT) ends.", 
      possible_visit_codes = "1700, 1711",
      visit_number = "V7",
      study_week = 7
      ),
    tibble(
      visit = "7w post-INT", 
      definition = "Visit seven weeks after the intervention (INT) ends.", 
      possible_visit_codes = "1900, 1911",
      visit_number = "V8",
      study_week = 9
      ),
    tibble(
      visit = "10w post-INT", 
      definition = "Visit ten weeks after the intervention (INT) ends.", 
      possible_visit_codes = "2120, 2121, 2122",
      visit_number = "V9",
      study_week = 12
    )
  ) |> 
  mutate(
    visit = visit |> fct_reorder(study_week)
  )

visit_dict |> 
  gt() |> 
  tab_header(
    title = "Visit codes and definitions"
  ) |> 
  cols_label(
    visit = "Visit",
    definition = "Definition",
    possible_visit_codes = "Possible visit codes",
    visit_number = "Visit number",
    study_week = "Study week"
  ) |> 
  cols_align(
    align = "left", columns = c(visit, definition, possible_visit_codes)
  )

Visit codes and definitions
Visit	Definition	Possible visit codes	Visit number	Study week
screening	Screening visit for which we have the most assay data. If two screening visits have the same number of assays, we take the last one.	0000, 0001, 0010, 0011	V0	-1
add. screening	If a participant has data for two screening visits, one is the "main" screening visit, and the other is labelled as an additional screening visit.	0000, 0001, 0010, 0011	V0b	-2
pre-MTZ	Visit before the metronidazole treatment (MTZ) starts.	1000, 1011	V1	0
post-MTZ	Visit after the metronidazole treatment (MTZ) ends.	1100	V2	1
post-INT	Visit after the intervention (INT) ends.	1200, 1211	V3	2
1w post-INT	Visit one week after the intervention (INT) ends.	1300	V4	3
2w post-INT	Visit two weeks after the intervention (INT) ends.	1400	V5	4
3w post-INT	Visit three weeks after the intervention (INT) ends.	1500, 1511, 1512	V6	5
5w post-INT	Visit five weeks after the intervention (INT) ends.	1700, 1711	V7	7
7w post-INT	Visit seven weeks after the intervention (INT) ends.	1900, 1911	V8	9
10w post-INT	Visit ten weeks after the intervention (INT) ends.	2120, 2121, 2122	V9	12

We first determine, for each participant, which is their main vs. their additional screening visit.

mae_coldata <- 
  mae_coldata |> 
  group_by(pid) |> 
  mutate(
    is_screening_visit = 
      case_when(
        visit_code %in% c("0000", "0001", "0010", "0011") ~ TRUE,
        TRUE ~ FALSE
      ),
    n_assays = ifelse(in_assays, str_split(assays, ", ") |> lengths(), 0),
    screening_visit_score = is_screening_visit * (n_assays + study_day/10)
  ) |> 
  arrange(-screening_visit_score) |> 
  mutate(
    screening_visit = 
      case_when(
        is_screening_visit & (row_number() == 1) ~ "main screening",
        is_screening_visit & (row_number() > 1) ~ "additional screening",
        TRUE ~ NA_character_
      )
  ) |> 
  ungroup() |> 
  select(-is_screening_visit, -n_assays, -screening_visit_score)

mae_coldata |> 
  dplyr::filter(screening_visit == "additional screening") |> 
  select(pid) |> distinct() |> 
  left_join(mae_coldata) |> 
  arrange(randomized |> desc(), pid, visit_code) |> 
  select(randomized, pid, visit_code, study_day, crf_plates, assays, screening_visit) |> 
  View()

mae_coldata |> 
  dplyr::filter(screening_visit == "additional screening") |> 
  select(pid) |> distinct() |> 
  left_join(mae_coldata) |> 
  arrange(randomized |> desc(), pid, visit_code) |> 
  select(randomized, pid, visit_code, study_day, crf_plates, assays, screening_visit) |> 
  dplyr::filter(randomized, (screening_visit == "main screening"), study_day == 0) |> 
  select(pid) |> distinct() |> 
  left_join(mae_coldata) |> 
  arrange(randomized |> desc(), pid, visit_code) |> 
  select(randomized, mITT, pid, visit_code, study_day, crf_plates, assays, screening_visit) |> 
  View()


# > check 068200020
# unclear - her main screening visit could be at day -3, but it might be at day 0, which would mean that her screening visit and pre-MTZ visit samples were collected on the same day
crf_raw$crf35 |> dplyr::filter(pid == "068200020") |> View()

mae_coldata |> 
  filter(!is.na(screening_visit)) |> 
  dplyr::count(screening_visit) 

# A tibble: 2 × 2
  screening_visit          n
  <chr>                <int>
1 additional screening    66
2 main screening         532

We next join the mae_coldata table with the visit_dict table to add the visit, visit_number, and study_week columns.

mae_coldata <- 
  mae_coldata |> 
  select(-any_of(c("PIPV", "visit", "visit_number", "study_week"))) |>
  left_join(
    visit_dict |> 
      mutate(visit_code = possible_visit_codes |> str_split(", ")) |>
      unnest(visit_code) |>
      mutate(
        screening_visit = 
          case_when(
            visit_number == "V0b" ~ "additional screening",
            visit_number == "V0" ~ "main screening",
            TRUE ~ NA_character_
            ),
        PIPV = visit_number != "V0b"
        ) |> 
      select(visit_code, screening_visit, PIPV, visit, visit_number, study_week)
  ) |> 
  mutate(PIPV = PIPV |> replace_na(FALSE)) |> 
  relocate(
    PIPV, visit, visit_number, study_week, .after = "visit_code"
  ) 

We check that study_days are unique for each visit_number at which assay samples were collected for each participant.

mae_coldata |> 
  group_by(pid, visit_number) |> 
  mutate(n_study_day = n_distinct(study_day[!is.na(assays)])) |>
  ungroup() |> 
  filter(PIPV, n_study_day > 1) |> 
  select(uid, pid, visit_code, study_day, assays) |> 
  group_by(pid) |> 
  gt(row_group_as_column = TRUE)

	uid	visit_code	study_day	assays
068200062	068200062_1500	1500	37	mg, qPCR, amplicon
	068200062_1511	1511	44	luminex, flow
068200087	068200087_1500	1500	40	mg, qPCR, amplicon, luminex
	068200087_1511	1511	43	flow

There are just two participants for which the dates don’t match and the different assays were collected at different dates. This may be important for integrative analyses later, so we leave as is for now.

Creating an assay to document specimen collection, and available data

• specimen_collection_swab (collected, not collected, unclear)
• specimen_collection_softcup (collected, not collected, unclear)
• specimen_collection_cytobrush (collected, not collected, unclear)
• mg
• qPCR
• amplicon
• luminex
• flow

sample_and_visit_info_assay <- 
  mae_coldata |> 
  select(uid, sample_type, in_CRF, in_assays, randomized) 

sample_and_visit_info_assay <- 
  sample_and_visit_info_assay |> 
  dplyr::left_join(
    visits |> select(uid, starts_with("specimen_collection")),
    by = join_by(uid)
  ) 

sample_and_visit_info_assay <-
  sample_and_visit_info_assay |>
  expand_grid(assay = assay_list$mae_assay_name) |>
  dplyr::left_join(
    available_samples |>
      select(uid, assay) |>
      mutate(value = TRUE),
    by = join_by(uid, assay)
  ) |>
  mutate(value = value |> replace_na(FALSE)) |>
  pivot_wider(names_from = assay, values_from = value)

# sample_and_visit_info_assay <- 
#   sample_and_visit_info_assay |> 
#   mutate(
#     mg_category = 
#       case_when(
#         !str_detect(sample_type, "Clinical sample") & mg ~ "Not a clinical sample",
#         is.na(randomized) & mg ~ "???",
#         !is.na(randomized) & !randomized & mg ~ "Additional sample from non-randomized participants",
#         !is.na(specimen_collection_swab) & (specimen_collection_swab > 0) & mg ~ "Expected data",
#         (is.na(specimen_collection_swab) | (specimen_collection_swab == 0)) & mg ~ "Unexpected data",
#         !is.na(specimen_collection_swab) & (specimen_collection_swab > 0) & !mg ~ "Missing data",
#         (is.na(specimen_collection_swab) | (specimen_collection_swab == 0)) & !mg ~ "Sample not collected",
#         TRUE ~ "ERROR"
#       ),
#     qPCR_category = 
#       case_when(
#         !str_detect(sample_type, "Clinical sample") & qPCR ~ "Not a clinical sample",
#         is.na(randomized) & qPCR ~ "???",
#         !is.na(randomized) & !randomized & qPCR ~ "Additional sample from non-randomized participants",
#         !is.na(specimen_collection_swab) & (specimen_collection_swab > 0) & qPCR ~ "Expected data",
#         (is.na(specimen_collection_swab) | (specimen_collection_swab == 0)) & qPCR ~ "Unexpected data",
#         !is.na(specimen_collection_swab) & (specimen_collection_swab > 0) & !qPCR ~ "Missing data",
#         (is.na(specimen_collection_swab) | (specimen_collection_swab == 0)) & !qPCR ~ "Sample not collected",
#         TRUE ~ "ERROR"
#       ),
#     amplicon_category = 
#       case_when(
#         !str_detect(sample_type, "Clinical sample") & amplicon ~ "Not a clinical sample",
#         is.na(randomized) & amplicon ~ "???",
#         !is.na(randomized) & !randomized & amplicon ~ "Additional sample from non-randomized participants",
#         !is.na(specimen_collection_swab) & (specimen_collection_swab > 0) & amplicon ~ "Expected data",
#         (is.na(specimen_collection_swab) | (specimen_collection_swab == 0)) & amplicon ~ "Unexpected data",
#         !is.na(specimen_collection_swab) & (specimen_collection_swab > 0) & !amplicon ~ "Missing data",
#         (is.na(specimen_collection_swab) | (specimen_collection_swab == 0)) & !amplicon ~ "Sample not collected",
#         TRUE ~ "ERROR"
#       ),
#     luminex_category = 
#       case_when(
#         !str_detect(sample_type, "Clinical sample") & luminex ~ "Not a clinical sample",
#         is.na(randomized) & luminex ~ "???",
#         !is.na(randomized) & !randomized & luminex ~ "Additional sample from non-randomized participants",
#         !is.na(specimen_collection_softcup) & (specimen_collection_softcup != "not collected") & luminex ~ "Expected data",
#         (is.na(specimen_collection_softcup) | (specimen_collection_softcup == "not collected")) & luminex ~ "Unexpected data",
#         !is.na(specimen_collection_softcup) & (specimen_collection_softcup == "collected") & !luminex ~ "Missing data",
#         (is.na(specimen_collection_softcup) | (specimen_collection_softcup == "not collected")) & !luminex ~ "Sample not collected",
#         TRUE ~ "ERROR"
#       )
#   )

# 
# The last 5 columns can take the following values:
# 
# - `expected` (swab collected and available data), 
# - `unexpected` (swab not collected but available data), 
# - `missing` (swab collected but no available data),
# - `maybe missing` (unclear if swab was collected and no available data),
# - `not collected` (swab not collected), 
# - `additional` (swab collected from a non-randomized participant), 
# - `NA` (not collected, no available data)
# 

se_sample_and_visit_info_assay <- 
  SummarizedExperiment(
    assays = list(
      sample_and_visit_info = 
        sample_and_visit_info_assay |> 
        select(-c(sample_type, in_CRF, in_assays, randomized)) |> 
        as.data.frame() |> 
        column_to_rownames("uid") |> 
        t()
    ),
    colData = 
      sample_and_visit_info_assay |> select(uid) |> 
      mutate(rownames = uid) |> as.data.frame() |> 
      column_to_rownames("rownames")
  )

Creating the MultiAssayExperiment object

SE_list <- 
  list(
    visit_and_sample_info = se_sample_and_visit_info_assay,
    mg = se_mg,
    qPCR = se_qPRC,
    amplicon = se_16S,
    luminex = se_luminex,
    flow = se_flow
  )


mae <- 
  MultiAssayExperiment::MultiAssayExperiment(
    experiments = MultiAssayExperiment::ExperimentList(SE_list),
    colData = 
      mae_coldata |> select(-in_CRF, -in_assays) |> 
      as.data.frame() |> mutate(rownames = uid) |> 
      column_to_rownames("rownames"),
    metadata = list(
      study = "VIBRANT",
      data_source = "actual study data (not simulated)",
      data_status = "partially QCed",
      date = today(),
      colData_dictionary = participants_variable_dictionary,
      crf_plates_description = crf_plates_dictionary,
      crf_data_raw = crf_raw,
      crf_data_clean = crf_clean, 
      participant_crfs_merged = participant_crfs_merged,
      visits_crfs_merged = visits_crfs_merged,
      exposures = exposures
    )
  )

mae

A MultiAssayExperiment object of 6 listed
 experiments with user-defined names and respective classes.
 Containing an ExperimentList class object of length 6:
 [1] visit_and_sample_info: SummarizedExperiment with 8 rows and 5462 columns
 [2] mg: SummarizedExperiment with 779 rows and 996 columns
 [3] qPCR: SummarizedExperiment with 16 rows and 1029 columns
 [4] amplicon: SummarizedExperiment with 817 rows and 4119 columns
 [5] luminex: SummarizedExperiment with 48 rows and 850 columns
 [6] flow: SummarizedExperiment with 11 rows and 356 columns
Functionality:
 experiments() - obtain the ExperimentList instance
 colData() - the primary/phenotype DataFrame
 sampleMap() - the sample coordination DataFrame
 `$`, `[`, `[[` - extract colData columns, subset, or experiment
 *Format() - convert into a long or wide DataFrame
 assays() - convert ExperimentList to a SimpleList of matrices
 exportClass() - save data to flat files

Available assays

mae_sample_map <- 
  mae@sampleMap |> as_tibble() |> dplyr::filter(assay %in% assay_list$mae_assay_name) |>
  select(uid = primary, assay) |> 
  arrange(uid) |> 
  dplyr::left_join(assay_list |> dplyr::rename(assay = mae_assay_name), by = join_by(assay)) |> 
  dplyr::right_join(
    mae@colData |> as.data.frame() |> as_tibble() |> 
      select(uid, pid, visit_code, PIPV, visit, visit_type, sample_type, location, randomized, arm), 
    by = join_by(uid)
    )

All samples

mae_sample_map |> 
  dplyr::filter(!is.na(assay)) |> 
  group_by(uid) |> mutate(n = n()) |> ungroup() |> 
  mutate(uid = uid |> fct_reorder(-n)) |>
  ggplot() +
  aes(x = uid, y = assay_label |> fct_rev(), fill = assay_label) +
  geom_tile() +
  facet_grid(. ~ sample_type, scales = "free_x", space = "free_x") +
  scale_x_discrete("Sample IDs", breaks = NULL) +
  ylab("") +
  scale_fill_brewer("Assay", type = "qual", palette = 7) + # 3
  theme(
    axis.text.x = element_text(angle = 45, hjust = 1),
    strip.text.x = element_text(angle = 90),
    legend.position = "bottom"
    )

Longitudinal availability for all visits

mae_sample_map |> 
  dplyr::filter(!is.na(assay)) |> 
  # filter(visit_code %in% c("0000", seq(1000, 1900, by = 100), "2120")) |> 
  ggplot() +
  aes(x = assay_label, y = pid, fill = assay_label) +
  geom_tile() +
  facet_grid(
    ifelse(randomized, "Randomized", "Not randomized") + location ~ visit_code, 
    scales = "free_y", space = "free_y"
    ) + # sample_category
  scale_y_discrete("Participants IDs") + #, breaks = "NULL")  +
  xlab("") +
  scale_fill_brewer("Assay", type = "qual", palette = 7) + # 3
  theme(
    axis.text.x = element_blank(), #element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0),
    strip.text.x = element_text(angle = 90),
    legend.position = "bottom"
    )

For the weekly visits of all participants

mae_sample_map |> 
  dplyr::filter(!is.na(assay)) |> 
  dplyr::filter(visit_type == "Clinic") |> 
  ggplot() +
  aes(x = assay_label, y = pid, fill = assay_label) +
  geom_tile() +
  facet_grid(
    location + ifelse(randomized, "Randomized", "Not randomized")  + arm  ~ visit_code, 
    scales = "free_y", space = "free_y"
    ) + # sample_category
  scale_y_discrete("Participants IDs") + #, breaks = "NULL")  +
  xlab("") +
  scale_fill_brewer("Assay", type = "qual", palette = 7) + # 3
  theme(
    axis.text.x = element_blank(), #element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0),
    strip.text.x = element_text(angle = 90),
    legend.position = "bottom"
    )

For the weekly visits of randomized participants

mae_sample_map |> 
  dplyr::filter(randomized) |> 
  dplyr::filter(!is.na(assay)) |> 
  dplyr::filter(visit_type == "Clinic") |> 
  ggplot() +
  aes(x = assay_label, y = pid, fill = assay_label) +
  geom_tile() +
  facet_grid(
    location + ifelse(randomized, "Randomized", "Not randomized")  + arm  ~ visit_code, 
    scales = "free_y", space = "free_y"
    ) + # sample_category
  scale_y_discrete("Participants IDs") + #, breaks = "NULL")  +
  xlab("") +
  scale_fill_brewer("Assay", type = "qual", palette = 7) + # 3
  theme(
    axis.text.x = element_blank(), #element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0),
    strip.text.x = element_text(angle = 90),
    legend.position = "bottom"
    )

mae_sample_map |> 
  dplyr::filter(randomized) |> 
  dplyr::filter(!is.na(assay)) |> 
  dplyr::filter(PIPV) |> 
  ggplot() +
  aes(x = assay_label, y = pid, fill = assay_label) +
  geom_tile() +
  facet_grid(
    location + arm  ~ visit, 
    scales = "free_y", space = "free_y"
    ) + # sample_category
  scale_y_discrete("Participants IDs") + #, breaks = "NULL")  +
  xlab("") +
  scale_fill_brewer("Assay", type = "qual", palette = 7) + # 3
  theme(
    axis.text.x = element_blank(), #element_text(angle = 45, hjust = 1),
    strip.text.y = element_text(angle = 0),
    strip.text.x = element_text(angle = 90),
    legend.position = "bottom"
    )

Subsetting and saving the MultiAssayExperiment objects

We save the “master” MAE (with all assays); but if needed for publication, we can subset the MAE to only include the assays/data of interest.

saveRDS(
  mae, 
  str_c(
    get_02_output_dir(),
    "mae_full_", today() |> str_remove_all("-"), ".rds"
  )
)